Fused pentacyclic polyethers

ABSTRACT

Disclosed are polycyclic polyether compounds of formula I and pharmaceutical compositions comprising such compounds. 
     
       
         
         
             
             
         
       
         
         
           
             wherein R, OR 1 , and R 2  are as defined herein. 
           
         
       
    
     Also disclosed are methods of regulating mucus clearance in a cell, and methods of treating decreased mucus clearance or mucociliary dysfunction.

This application claims priority from U.S. Provisional PatentApplication No. 60/504,669, which was filed Sep. 19, 2003.

FIELD OF THE INVENTION

The invention relates to polycyclic polyether compounds, pharmaceuticalcompositions comprising the compounds, and methods of treating diseasesusing the compounds and pharmaceutical compositions. More specifically,the invention relates to a naturally occurring polycyclic polyethercompound, “brevenal”, isolated from the marine dinoflagellate K. brevis,and derivatives thereof. It also relates to pharmaceutical compositionsand methods of treating brevetoxin and ciguatoxin poisoning, anddiseases that are characterized by decreased mucus clearance andmucociliary dysfunction, comprising the compounds and pharmaceuticalcompositions.

BACKGROUND

Decreased mucus clearance is a pathologic characteristic of diseasessuch as cystic fibrosis; chronic obstructive airway disease (also knownas chronic obstructive pulmonary disease (COPD)) and asthma. Impairedmucus clearance can also contribute to increased incidence of pulmonaryinfections and airway obstruction. In particular, cystic fibrosis ischaracterized by abnormal functioning of the airway epithelial cells.Cystic fibrosis (or “CF”) is caused by a defective gene that codes for aNa⁺/Cl⁻ transporter present on the surface of the epithelial cells thatline the conducting airways of the lung and other organs. Hundreds ofmutations have been identified in this gene, all of which result indefective transport of sodium and chloride by epithelial cells. Cysticfibrosis (CF) is the most common autosomal recessive genetic disease inCaucasians causing premature death in the United States. It is caused bymutations in chromosome 7, which code for the cystic fibrosistransmembrane conductance regulator (CFTR). The CFTR encodes for anapical membrane epithelial protein that function as both acCAMP-regulated chloride channel and a regulator of the epithelialsodium channel. Defects or absence of the CFTR observed in CF patientscan be seen as changes in cilia ultrastructure, sodium and chloride iontransport, and water transport across airway epithelial cells. Thesechanges can result in thickened mucous and decreased mucociliaryclearance leading to airway infections. This suggests that CFTR innormal lung tissue may regulate the ENaC by down regulating itsconductance of sodium ions across the airway epithelium and decreasingwater transport into the cell resulting in less viscous mucous andfaster mucociliary clearance.

Current treatment for CF has focused on several different therapies butthe most effective treatments to date are compounds that change theviscosity of mucous and treat pulmonary infections that arise whenbacteria are trapped in the thickened mucous. Therapies to increasemucociliary clearance generally target two different strategies. Thefirst is to regulate sodium absorption into the apical epithelial cellsusing sodium channel blockers such as amiloride and its derivatives. Bydecreasing sodium absorption, fluid transport into the epithelial cellsis limited and the surface liquid volume is normalized.

The second strategy is to utilize compounds targeting purinergicreceptors (i.e. UTP and INS37217) which activate chloride secretion inairway epithelial cells which in turn decreases sodium absorption andincreases surface liquid volume. Purinergic receptors are also thoughtto regulate mucin secretion and to be involved in activation of ciliarybeating. By increasing the ciliary beat frequency, mucous transportwould be increased which would clear bacteria and other particles fromthe lungs more rapidly.

These observations indicate that activation of sodium channels can leadto defects in mucus clearance and bronchoconstriction, both of which areassociated with airway diseases, including CF.

If activation of voltage gated sodium channels contributes to lungdiseases, then effective modulation of voltage gated sodium channels canbe useful in alleviating airway pathologies associated with mucociliarydysfunction, such as asthma, chronic obstructive pulmonary diseases,pulmonary infection (e.g., pneumonia, Pseudomonas), and cystic fibrosis.Thus, there is a need for active agents that can modulate watertransport across the apical membranes of epithelial cells, thesecompounds would be are useful in the regulation of mucus clearance, aswell as treatment or prevention of conditions or diseases associatedwith mucociliary dysfunction.

Florida red tides are known to have adverse effects on both marine lifeand humans. These tides have been linked to large fish kills, marinemammal mortality, and even human illnesses. Human illnesses caused byred tides include respiratory irritation through contact or inhalationand neurotoxic shellfish poisoning (NSP) from consumption of exposed orcontaminated seafood (Purkerson-Parker, et al., Chemistry and Biology,2000, 7: 385–393; Baden, D. G., et al., Toxicon, 1982; 20(5):929–932;Baden, D. G., et al., Int. Rev. Cytol., 1983; 82:99–150). Symptoms ofNSP include nausea, vomiting, diarrhea, and bronchoconstriction(Purkerson-Parker, et al. 2000). The causative agent in the red tideorganisms has been isolated and identified as brevetoxin.

Ciguatera fish poisoning (CFP) is a form of human poisoning caused bythe consumption of subtropical and tropical marine fish that haveaccumulated naturally occurring toxins through their diet. The toxinsare known to originate from several dinoflagellate (algae) species thatare common to ciguatera endemic regions in the lower latitudes. Marinefish most commonly implicated in ciguatera fish poisoning includegroupers, barracudas, snappers, jacks, mackerel, and triggerfish. Manyother species of warm-water fish can harbor ciguatera toxins. Theoccurrence of toxic fish is sporadic, and not all fish of a givenspecies or from a given locality will be toxic.

Initial signs of poisoning occur within six hours after consumption oftoxic fish and typically include a combination of gastrointestinal(e.g., nausea, vomiting, and diarrhea), neurological (e.g., intensifiedparesthesia, arthralgia, myalgia, headache, temperature sensory reversaland acute sensitivity to temperature extremes, vertigo, and muscularweakness), and cardiovascular disorders (e.g., arrhythmia, bradycardiaor tachycardia, and reduced blood pressure). Symptoms defined withinthese general categories vary with the geographic origin of toxic fish.Diagnosis of CFP remains unsatisfactory and is typically based onpatient symptoms and recent dietary history.

Ciguatera poisoning is usually self-limiting, and signs of poisoningoften subside within several days from onset. However, in severe casesthe neurological symptoms are known to persist from weeks to months. Ina few isolated cases neurological symptoms have persisted for severalyears, and in other cases recovered patients have experienced recurrenceof neurological symptoms months to years after recovery. Such relapsesare most often associated with changes in dietary habits or withconsumption of alcohol. There is a low incidence of death resulting fromrespiratory and cardiovascular failure.

Current treatments for ciguatera fish poisoning are far fromsatisfactory. Typically intravenous administration of mannitol is usedbut is normally only effective if it is used in the first 48–72 hours ofexposure. The treatment of chronic CFP is usually symptomatic.

CFP and NSP are thought to be induced via binding at a common receptorsite on voltage gated sodium channels known as site 5. Binding bybrevetoxins or ciguatoxin at site 5 results in massive influx in sodiumions at normal resting potential.

Thus, there is a need for active agents that can act as antagonists forbinding of brevetoxins or ciguatoxin to voltage gated sodium channels,which are useful in alleviating the neurological and gastrointestinaleffects in persons affected by NSP and CFP.

SUMMARY OF THE INVENTION

We have discovered a fused pentacyclic polyether compound havingactivity as a brevetoxin antagonist. This compound, Brevenal, isisolated from purified from native sources, such as K. brevis, and otherred tide organisms. Thus, in one aspect, the invention provides thecompound Brevenal which can be represented by the following formula:

In a broad aspect, the invention provides compounds of Formula I:

and pharmaceutically acceptable salts thereof, wherein

-   R is C₁–C₁₂ alkyl, C₂–C₁₂ alkenyl, C₁–C₁₂ alkyl esters, C₁–C₁₂ alkyl    amides, C₄–C₁₂ alkenyl esters, C₁–C₁₂ alkylaryl esters, C₄–C₁₂    alkenylaryl esters, C₄–C₁₂ alkenyl amides, C₁–C₁₂ alkoxy,    formylC₁–C₁₂alkyl, formylC₂–C₁₂alkenyl, alkanoylC₁–C₁₂alkyl,    alkanoylC₂–C₁₂alkenyl, carboxyC₁–C₁₂alkyl, carboxyC₂–C₁₂alkenyl,    wherein the alkyl and alkenyl groups are optionally substituted with    1–6 substituent groups selected from the group consisting of: C₃–C₁₂    cycloalkyl, C₃–C₁₂ heterocyclyl, aryl, heteroaryl, C₁–C₆ alkyl,    C₁–C₆ alkoxy, halogen, C₂–C₆ alkenyl, OH, nucleosides, nucleotides,    purines, pyrimidines, aromatic esters, aryl esters, cycloalkyl    esters, cycloalkenyl esters, purines, or pyrimidines;-   OR₁ is OH or —O(CO)CH₃; and-   R₂ is —CH═CHCH═CH₂, —CH₂-phenyl, or —CH₂-pyridyl, wherein the phenyl    and pyridyl groups are optionally substituted at each substitutable    position with a group that is independently C₁–C₆ alkyl, C₁–C₆    alkoxy, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, halogen,    —CO₂H, C₁–C₆ alkoxycarbonyl, —C(O)NH₂, —C(O)NH(C₁–C₆ alkyl), or    —C(O)N(C₁–C₆ alkyl) (C₁–C₆ alkyl) or-   pharmaceutically acceptable salts, solvates, esters, amides,    hydrates, or combinations thereof.

The compounds of the invention have activity as antagonists ofbrevetoxins and are therefore useful in treating brevetoxin andciguatoxin poisoning.

The present invention also provides compounds of Formula I where

-   R is

wherein

Hal is chloro, fluoro, iodo, or bromo;

R₃ is selected from H, OH, NH₂, halogen, and NO₂, and

Y is selected from CH, N, O, and S.

The invention also relates to pharmaceutical compositions comprising acompound of formula I, or a pharmaceutically acceptable salt, hydrate orsolvate thereof, in combination with a pharmaceutically acceptablecarrier, excipient, solvent, adjuvant or diluent.

The compounds of the invention can also act as antagonists for the classof compounds known as the brevetoxins and ciguatoxin.

The invention further relates to methods of regulating mucus clearancevelocity and treating conditions or diseases associated with decreasedmucus clearance and mucociliary dysfunction in a subject comprisingadministering to a subject a compound of formula I or a pharmaceuticallyacceptable salt, solvate, or hydrate thereof.

The invention also provides methods for treating brevetoxin andciguatoxin poisoning, comprising administering to a subject a compoundof the invention or a pharmaceutically acceptable salt, solvate, orhydrate thereof, in an amount effective to treat brevetoxin orciguatoxin poisoning. This method of treating brevetoxin and ciguatoxinpoisoning can help prevent, treat, reduce the severity of, or delay theonset or progression of symptoms and disease states associated withbrevetoxin and ciguatoxin poisoning.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the effect of brevenal concentrations (3, 10, and 100pg/mL) on PbTx-3 induced airway constriction in sheep.

FIG. 2 illustrates the effect of brevenal concentrations (3, 10, and 100pg/mL) on PbTx-2 induced airway constriction in sheep.

FIG. 3 illustrates the effect of brevenal (10 pg/mL) on hemi-brevetoxininduced airway constriction in sheep.

FIG. 4 illustrates the effect of β-naphthoyl-PbTx-3 and brevenal (100pg/mL) on PbTx-3 induced reduction of tracheal mucus velocity (TMV) insheep.

FIG. 5 illustrates the effect of PbTx-3 and PbTx-2 (CONC.10 pg/mL) ontracheal mucus velocity (TMV) in sheep.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by one of skill in the artto which this invention belongs.

All patents and publications referred to herein are hereby incorporatedby reference for all purposes.

A “therapeutically effective” amount is defined as an amount effectiveto reduce or lessen at least one symptom of the disease being treated orto reduce or delay onset of one or more clinical markers or symptoms ofthe disease.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to acomposition containing “a compound” includes a mixture of two or morecompounds. It should also be noted that the term “or” is generallyemployed in its sense including “and/or” unless the content clearlydictates otherwise.

By “alkyl” and “C₁–C₆ alkyl” in the present invention is meant straightor branched chain alkyl groups having 1–6 carbon atoms, such as, methyl,ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl,2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and3-methylpentyl. It is understood that in cases where an alkyl chain of asubstituent (e.g. of an alkyl, alkoxy or alkenyl group) is shorter orlonger than 6 carbons, it will be so indicated in the second “C” as, forexample, “C₁–C₁₀” indicates a maximum of 10 carbons.

By the term “halogen” in the present invention is meant fluorine,bromine, chlorine, and iodine.

“Alkenyl” and “C₂–C_(n) alkenyl” means straight and branched hydrocarbongroups having from 2 to n carbon atoms and from one to four double bondsand includes, for example, ethenyl, propenyl, 1-but-3-enyl,1-pent-3-enyl, 1-hex-5-enyl, (3E,5E)-4,5-dimethyldeca-3,5-diene and thelike.

As used herein, the term “cycloalkyl” refers to saturated carbocyclicgroups having three to twelve carbon atoms. The cycloalkyl can bemonocyclic, or a polycyclic fused system. Examples of such groupsinclude cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Thecycloalkyl groups herein are unsubstituted or, as specified, substitutedin one or more substitutable positions with various groups. For example,such cycloalkyl groups may be optionally substituted with, for example,C₁–C₆ alkyl, C₁–C₆ alkoxy, halogen, hydroxy, cyano, nitro, amino,mono(C₁–C₆)alkylamino, di(C₁–C₆)alkylamino, C₂–C₆alkenyl, C₂–C₆alkynyl,C₁–C₆ haloalkyl, C₁–C₆ haloalkoxy, amino(C₁–C₆)alkyl,mono(C₁–C₆)alkylamino(C₁–C₆)alkyl or di (C₁–C₆)alkylamino(C₁–C₆)alkyl.

By “aryl” is meant an aromatic carbocyclic group having a single ring(e.g., phenyl), multiple rings (e.g., biphenyl), or multiple condensedrings in which at least one is aromatic, (e.g.,1,2,3,4-tetrahydronaphthyl, naphthyl), which is optionally mono-, di-,or trisubstituted. Preferred aryl groups of the present invention arephenyl, 1-naphthyl, 2-naphthyl, indanyl, indenyl, dihydronaphthyl,tetralinyl or 6,7,8,9-tetrahydro-5H-benzo[a]cycloheptenyl. The arylgroups herein are unsubstituted or, as specified, substituted in one ormore substitutable positions with various groups. Preferred aryl groupsare optionally substituted with C₁–C₆ alkyl, C₁–C₆ alkoxy, halogen,hydroxy, cyano, nitro, amino, mono(C₁–C₆)alkylamino,di(C₁–C₆)alkylamino, C₂–C₆alkenyl, C₂–C₆alkynyl, C₁–C₆ haloalkyl, C₁–C₆haloalkoxy, amino(C₁–C₆)alkyl, mono(C₁–C₆)alkylamino(C₁–C₆)alkyl ordi(C₁–C₆)alkylamino(C₁–C₆)alkyl. Preferred aryl groups are phenyl andnaphthyl, each of which is optionally substituted as described above.

By “heteroaryl” is meant one or more aromatic ring systems of 5-, 6-, or7-membered rings which includes fused ring systems of 9–11 atomscontaining at least one and up to four heteroatoms selected fromnitrogen, oxygen, or sulfur. Preferred heteroaryl groups of the presentinvention include pyridinyl, pyrimidinyl, quinolinyl, benzothienyl,indolyl, indolinyl, pryidazinyl, pyrazinyl, isoindolyl, isoquinolyl,quinazolinyl, quinoxalinyl, phthalazinyl, imidazolyl, isoxazolyl,pyrazolyl, oxazolyl, thiazolyl, indolizinyl, indazolyl, benzothiazolyl,benzimidazolyl, benzofuranyl, furanyl, thienyl, pyrrolyl, oxadiazolyl,thiadiazolyl, triazolyl, tetrazolyl, oxazolopyridinyl, imidazopyridinyl,isothiazolyl, naphthyridinyl, cinnolinyl, carbazolyl, beta-carbolinyl,isochromanyl, chromanyl, tetrahydroisoquinolinyl, isoindolinyl,isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isobenzothienyl,benzoxazolyl, pyridopyridinyl, benzotetrahydrofuranyl,benzotetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, phenoxazinyl,phenothiazinyl, pteridinyl, benzothiazolyl, imidazopyridinyl,imidazothiazolyl, dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl,dihydrobenzisothiazinyl, benzopyranyl, benzothiopyranyl, coumarinyl,isocoumarinyl, chromonyl, chromanonyl, pyridinyl-N-oxide,tetrahydroquinolinyl, dihydroquinolinyl, dihydroquinolinonyl,dihydroisoquinolinonyl, dihydrocoumarinyl, dihydroisocoumarinyl,isoindolinonyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl N-oxide,pyrimidinyl N-oxide, pyridazinyl N-oxide, pyrazinyl N-oxide, quinolinylN-oxide, indolyl N-oxide, indolinyl N-oxide, isoquinolyl N-oxide,quinazolinyl N-oxide, quinoxalinyl N-oxide, phthalazinyl N-oxide,imidazolyl N-oxide, isoxazolyl N-oxide, oxazolyl N-oxide, thiazolylN-oxide, indolizinyl N-oxide, indazolyl N-oxide, benzothiazolyl N-oxide,benzimidazolyl N-oxide, pyrrolyl N-oxide, oxadiazolyl N-oxide,thiadiazolyl N-oxide, triazolyl N-oxide, tetrazolyl N-oxide,benzothiopyranyl S-oxide, benzothiopyranyl S,S-dioxide. More preferredheteroaryl groups include oxazolyl, isoxazolyl, pyridyl, pyrimidyl,pyridazinyl, and pyrazinyl. The heteroaryl groups herein areunsubstituted or, as specified, substituted in one or more substitutablepositions with various groups. Preferred heteroaryl groups areoptionally substituted with C₁–C₆ alkyl, C₁–C₆ alkoxy, halogen, hydroxy,cyano, nitro, amino, mono(C₁–C₆)alkylamino, di(C₁–C₆)alkylamino,C₂–C₆alkenyl, C₂–C₆alkynyl, C₁–C₆ haloalkyl, C₁–C₆ haloalkoxy,amino(C₁–C₆)alkyl, mono(C₁–C₆)alkylamino(C₁–C₆)alkyl ordi(C₁–C₆)alkylamino(C₁–C₆)alkyl.

By “heterocycle”, “heterocycloalkyl” or “heterocyclyl” is meant one ormore carbocyclic ring systems of 4-, 5-, 6-, or 7-membered rings whichincludes fused ring systems of 9–11 atoms containing at least one and upto four heteroatoms selected from nitrogen, oxygen, or sulfur. Preferredheterocycles of the present invention include morpholinyl,thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S,S-dioxide,piperazinyl, homopiperazinyl, pyrrolidinyl, pyrrolinyl,tetrahydropyranyl, piperidinyl, tetrahydrofuranyl,pyrimidine-2,4(1H,3H)-dione, 1H-benzo[d]imidazol-2(3H)-onetetrahydrothienyl, homopiperidinyl, homomorpholinyl,homothiomorpholinyl, homothiomorpholinyl S,S-dioxide, oxazolidinonyl,dihydropyrazolyl, dihydropyrrolyl, dihydropyrazinyl, dihydropyridinyl,dihydropyrimidinyl, dihydrofuryl, dihydropyranyl, tetrahydrothienylS-oxide, tetrahydrothienyl S,S-dioxide and homothiomorpholinyl S-oxide.More preferred heterocyclic groups include1H-benzo[d]imidazol-2(3H)-onyl, pyrimidine-2,4(1H,3H)-dionyl,piperidinyl, pyrrolidinyl and piperazinyl. The heterocycle groups hereinare unsubstituted or, as specified, substituted in one or moresubstitutable positions with various groups. Preferred heterocyclegroups are optionally substituted withC₁–C₆ alkyl, C₁–C₆ alkoxy,halogen, hydroxy, cyano, nitro, amino, mono(C₁–C₆)alkylamino,di(C₁–C₆)alkylamino, C₂–C₆alkenyl, C₂–C₆alkynyl, C₁–C₆ haloalkyl, C₁–C₆haloalkoxy, amino(C₁–C₆)alkyl, mono(C₁–C₆)alkylamino(C₁–C₆)alkyl,di(C₁–C₆)alkylamino(C₁–C₆)alkyl or ═O.

As used herein, the term “arylester” encompasses aryloxycarbonyl andarylcarbonyloxy groups.

As used herein, the term “alkylester” encompasses alkyloxycarbonyl andalkylcarbonyloxy groups. As used herein, alkylcarbonyl carries the samemeaning as alkanoyl.

As used herein, the term “alkylamide” encompasses alkylaminocarbonylgroups, dialkylcarbonyl groups, and alkanoylamino groups.

As used herein, the term “alkenylamide” encompasses alkenylaminocarbonylgroups, dialkenylcarbonyl groups, and alkenylcarbonylamino groups.

As used herein, the term “alkenylester” encompasses alkenyloxycarbonyland alkenylcarbonyloxy groups.

The term alkylarylester as used herein refers to alkyloxycarbonyl andakanoyloxy groups in which the alkyl portion carries an aryl orheteroaryl group.

The term alkenylarylester as used herein refers to alkenyloxycarbonyland alkenylcarbonyloxy groups in which the alkenyl portion carries anaryl or heteroaryl group.

The phrase “regulating mucous clearance” encompasses “controlling,promoting and/or influencing mucous clearance.”

As used herein, the terms “treatment” and “treating” encompassprophylactic administration of the compound or a pharmaceuticalcomposition comprising the compound (“prophylaxis”) as well as remedialtherapy to reduce or eliminate a disease or disorder mentioned herein.Prophylactic administration is intended for preventing disorders orpreventing the recurrence of disorders and may be used to treat asubject that is at risk of having or suffering from one or moredisorders mentioned herein. Thus, as used herein, the term “treatment”,or a derivative thereof, contemplates partial or complete inhibition ofthe stated disease state, when an active ingredient of the invention isadministered prophylactically or following the onset of the diseasestate for which such active ingredient of the is administered.“Prophylaxis” refers to administration of the active ingredient(s) to amammal to protect the mammal from any of the disorders set forth herein,as well as others.

As used herein, the term “subject” encompasses animals, includingmammals and fish. Preferably the term refers to mammals such as humans,cattle and horses, more preferably to humans and domestic animals suchas cats, dogs, and horses, and most preferably to humans.

In one aspect, the present invention relates to compounds, orpharmaceutically acceptable salts thereof, of Formula (I):

wherein

-   R is C₁–C₁₂ alkyl, C₂–C₁₂ alkenyl, C₁–C₁₂ alkyl esters, C₁–C₁₂ alkyl    amides, C₄–C₁₂ alkenyl esters, C₁–C₁₂ alkylaryl esters, C₄–C₁₂    alkenylaryl esters, C₄–C₁₂ alkenyl amides, C₁–C₁₂ alkoxy,    formylC₁–C₁₂alkyl, formylC₂–C₁₂alkenyl, alkanoylC₁–C₁₂alkyl,    alkanoylC₂–C₁₂alkenyl, carboxyC₁–C₁₂alkyl, carboxyC₂–C₁₂alkenyl,    wherein the alkyl and alkenyl groups are optionally substituted with    1–6 substituent groups selected from the group consisting of: C₃–C₁₂    cycloalkyl, C₃–C₁₂ heterocyclyl, aryl, heteroaryl, C₁–C₆ alkyl,    C₁–C₆ alkoxy, halogen, C₂–C₆ alkenyl, OH, nucleosides, nucleotides,    purines, pyrimidines, aromatic esters, aryl esters, cycloalkyl    esters, cycloalkenyl esters, purines, or pyrimidines;-   OR₁ is OH or —O(CO)CH₃; and-   R₂ is —CH═CHCH═CH₂, —CH₂-phenyl, or —CH₂-pyridyl, wherein the phenyl    and pyridyl groups are optionally substituted with 1, 2, or 3 groups    that are independently C₁–C₆ alkyl, C₁–C₆ alkoxy, haloalkyl,    haloalkoxy, hydroxy, hydroxyalkyl, halogen, —CO₂H, C₁–C₆    alkoxycarbonyl, —C(O)NH₂, —C(O)NH(C₁–C₆ alkyl), or —C(O)N(C₁–C₆    alkyl) (C₁–C₆ alkyl).

In another broad aspect, R₁ and R₂ are as defined above and R is alkyl,halogen, alkenyl, cycloalkyl, aryl, heteroaryl, heterocycle,heterocycloalkyl or heterocyclyl.

In yet another aspect, R is C₆–C₁₂ alkyl, C₆–C₁₂ alkyl esters, C₆–C₁₂alkyl amides, C₆–C₁₂ alkylphenyl esters, C₁–C₆ alkoxy,formylC₆–C₁₂alkyl, alkanoylC₆–C₁₂alkyl, carboxyC₆–C₁₂alkyl, wherein thealkyl portions are optionally substituted with 1–4 substituent groupsselected from the group consisting of: C₃–C₆ cycloalkyl, C₅–C₆heterocyclyl, penyl, naphthyl, pyridyl, pyrmidyl, pyridazinyl,pyrazinyl, furanyl, thienyl, quinolinyl, indolyl, C₁–C₆ alkyl, C₁–C₆alkoxy, halogen, C₂–C₆ alkenyl, OH, nucleosides, nucleotides, purines,pyrimidines, phenyl esters, cycloalkyl esters, cycloalkenyl esters,purines, and pyrimidines, wherein each of the cyclic substituents on Ris further optionally substituted with up to 5 groups that areindependently C₁–C₆ alkyl, C₁–C₆ alkoxy, halogen, haloalkyl, haloalkoxy,hydroxy, hydroxy C₁–C₆ alkyl, C₁–C₄ alkoxy C₁–C₆ alkyl, CO₂H, C(O)NH₂,C(O)NH(C₁–C₆ alkyl), or C(O)N(C₁–C₆ alkyl) (C₁–C₆ alkyl).

In yet another aspect, R is C₆–C₁₂ alkenyl, C₆–C₁₂ alkenyl esters,C₆–C₁₂ alkenylphenyl esters, C₆–C₁₂ alkenyl amides, formylC₆–C₁₂alkenyl,alkanoylC₆–C₁₂alkyl, alkanoylC₆–C₁₂alkenyl, carboxyC₆–C₁₂alkyl,carboxyC₆–C₁₂alkenyl, wherein the alkenyl groups are optionallysubstituted with 1–4 substituent groups selected from the groupconsisting of: C₃–C₆ cycloalkyl, C₅–C₆ heterocyclyl, penyl, naphthyl,pyridyl, pyrmidyl, pyridazinyl, pyrazinyl, furanyl, thienyl, quinolinyl,indolyl, C₁–C₆ alkyl, C₁–C₆ alkoxy, halogen, C₂–C₆ alkenyl, OH,nucleosides, nucleotides, purines, pyrimidines, phenyl esters,cycloalkyl esters, cycloalkenyl esters, purines, and pyrimidines,wherein each of the cyclic substituents on R is further optionallysubstituted with up to 5 groups that are independently C₁–C₆ alkyl,C₁–C₆ alkoxy, halogen, haloalkyl, haloalkoxy, hydroxy, hydroxy C₁–C₆alkyl, C₁–C₄ alkoxy C₁–C₆ alkyl, CO₂H, C(O)NH₂, C(O)NH(C₁–C₆ alkyl), orC(O)N(C₁–C₆ alkyl) (C₁–C₆ alkyl).

In still another aspect, OR₁ is OH.

In yet still another aspect, R₂ is —CH═CHCH═CH₂, —CH₂-phenyl, or—CH₂-pyridyl, wherein the phenyl and pyridyl groups are optionallysubstituted with 1, 2, or 3 groups that are independently C₁–C₆ alkyl,C₁–C₆ alkoxy, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, halogen,—CO₂H, C₁–C₆ alkoxycarbonyl, —C(O)NH₂, —C(O)NH(C₁–C₆ alkyl), or—C(O)N(C₁–C₆ alkyl) (C₁–C₆ alkyl).

In another aspect, R is C₆–C₁₂ alkenyl substituted with 1, 2, or 3groups that are independently C₁–C₄ alkoxy, halogen, —CHO, —OCH₂CH₂O—,—OCH₂CH₂CH₂O— or OH.

In yet another aspect, R is C₈–C₁₂ alkenyl substituted with 1, 2, or 3groups that are independently C₁–C₄ alkoxy, halogen, —CHO, —OCH₂CH₂O—,—OCH₂CH₂CH₂O— or OH.

In still yet another aspect, R is C₉–C₁₀ alkenyl substituted with 1, 2,or 3 groups that are independently C₁–C₄ alkoxy, —CHO, —OCH₂CH₂O—,—OCH₂CH₂CH₂O— or OH. In one aspect, when R is substituted with two C₁–C₄alkoxy groups, they are the same. Still more preferably, when the twoC₁–C₄ alkoxy groups are the same, they are attached to the same carbon,thereby forming an acetal.

In still another aspect, R is

In yet another aspect, R is

In still another aspect, R is

In another aspect, R is

In another embodiment of this aspect, the compound is of Formula (I),wherein R is

wherein

Hal is chloro, fluoro, iodo, or bromo;

R₃ is selected from H, OH, NH₂, halogen, and NO₂, and

Y is selected from CH, N, O, and S.

In another aspect, R₂ is —CH═CHCH═CH₂.

In a preferred embodiment, R₂ in the compound of Formula (I) is—CH═CHCH═CH₂ in cis conformation.

In a preferred embodiment, R₂ in the compound of Formula (I) is—CH═CHCH═CH₂ in cis conformation, and R is a C₂–C₁₂ alkenyl comprising adiene.

In a more preferred embodiment, R₂ in the compound of Formula (I) is—CH═CHCH═CH₂ in cis conformation, and R is a C₂–C₁₂ alkenyl comprising adiene, in trans conformation.

In another embodiment, R₂ in the compound of Formula (I) is CH═CHCH═CH₂in the trans conformation, and R is a C₈–C₁₂ alkenyl comprising a diene,in trans conformation.

In another embodiment, R₂ is —CH₂-phenyl, or —CH₂-pyridyl, wherein thephenyl and pyridyl groups are optionally substituted with 1, 2, or 3groups that are independently C₁–C₆ alkyl, C₁–C₆ alkoxy, CF₃, OCF₃,hydroxy, hydroxy C₁–C₆ alkyl, halogen, —CO₂H, C₁–C₆ alkoxycarbonyl,—C(O)NH₂, —C(O)NH(C₁–C₆ alkyl), or —C(O)N(C₁–C₆ alkyl) (C₁–C₆ alkyl)

In another embodiment, R₂ is —CH═CHCH═CH₂. In still another embodiment,R₂ is

In yet another embodiment, R₂ is

In yet another embodiment,

-   R is

and

-   R₂ is

In another embodiment R and R₂ are as defined above and R₁ is H.

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In another preferred embodiment, the compound of Formula (I) is:

In one aspect of this preferred embodiment, the compound of Formula (I)is:

In another preferred embodiment, the compound of Formula (I) is:

In one aspect of this preferred embodiment, the compound of Formula (I)is:

In another preferred embodiment, the compound of Formula (I) is:

In one aspect of this preferred embodiment, the compound of Formula (I)is:

In another preferred embodiment, the compound of Formula (I) is:

The compounds of Formula (I) may have asymmetric centers and occur asracemates, racemic mixtures and as individual diastereomers, orenantiomers. All isomeric forms are included within the scope of thepresent invention.

In a preferred aspect, the compounds of the invention are based on thebrevenal core, i.e., each stereocenter in the compounds of formula (I)have the same configuration as the stereocenters in brevenal.

In another aspect, the invention relates to pharmaceutical compositionscomprising a compound of formula I or a pharmaceutically acceptablesalt, solvate, or hydrate thereof, and a pharmaceutically acceptablecarrier, excipient, solvent, adjuvant or diluent.

In another aspect, the invention provides methods for regulating mucusclearance comprising administering to a subject, or contacting a cellwith, a compound, salt, solvate or hydrate of the invention, or apharmaceutical composition comprising a compound, salt, hydrate, orsolvate of the invention, in an amount effective to regulate mucusclearance in the subject or cell.

As used herein decreases in mucus clearance or mucociliary dysfunctionare generally measured by tracheal mucus transport (TMV), a surrogatemarker for whole lung clearance.

In another aspect, the invention provides methods for treatingconditions or diseases related to, or associated with, decreased mucusclearance comprising administering to a subject a compound of theinvention, or pharmaceutical composition comprising a compound of theinvention or a pharmaceutically acceptable salt, hydrate, ester, amidesolvate, or mixtures thereof, in an amount effective to treat thecondition or disease. This method of treating conditions or diseasesassociated with decreased mucus clearance can help prevent, treat,reduce the severity of, or delay the onset or progression of symptomsand disease states associated with decreased mucus clearance. Suchconditions or diseases include the non-limiting examples of chronicobstructive airway disease (also known as chronic obstructive pulmonarydisease (COPD)); asthma; cystic fibrosis, bronchoconstriction, and otherpulmonary diseases; including pulmonary infections, such as thenon-limiting examples pneumonia, Pseudomonas, and bronchitis; and cysticfibrosis.

In one embodiment, the method of treatment can be used to treat chronicobstructive pulmonary diseases, such as emphysema, pulmonary fibrosis,and/or smokers cough.

In one embodiment, the method of treatment can be used to treat asthma.

In one embodiment, the method of treatment can be used to treatpulmonary disease.

In one embodment, the method of treatment can be used to treat pulmonaryinfection, including, but not limited to, pneumonia, or Pseudomonas.

In one embodiment the method of treatment can be used to treat poisoningby brevetoxins or ciguatoxin, and the symptoms thereof

In a preferred embodiment, the method of treatment can be used to wherethe disease is cystic fibrosis.

In another aspect, the invention provides methods for treating thesymptoms related to conditions or diseases associated with decreasemucus clearance or mucociliary dysfunction, comprising administering toa subject in need of such treatment, a therapeutically effective amountof a compound of the invention, or a pharmaceutically acceptable salt,hydrate, or solvate thereof.

In an embodiment of this aspect the methods of the invention can be usedto treat diseases associated with mucus membranes, including suchdiseases involving pancreas, intestines, kidneys, fallopian tubes,and/or vas deferens.

In another embodiment of this aspect, the method can optionallycomprise, in combination with a compound of Formula (I) or apharmaceutically acceptable salt, solvate, or hydrate thereof, aneffective amount of a compound known to be useful for the treatment ofconditions or diseases associated with decreased mucus clearance. Themethods of the invention can optionally comprise additional therapeuticregimen such as supportive or adjuvant therapy.

In one embodiment of the methods of the invention, the subject is ananimal. More preferably, the animal is a mammal. Still more preferably,the mammal is a human.

In another embodiment, the animal is a companion animal, such as, forexample, a horse, dog or cat.

The therapeutically effective amounts of the compounds of the inventionsuitable for the methods are generally from about 0.1 pg/day to about1000 mg/day. The therapeutically effective amounts will vary accordingto various parameters including, for example, the particular therapeuticuse and physical characteristics of the subject/patient, and are wellwithin the knowledge of those skilled in the art.

In a preferred aspect, the therapeutically effective amount for oraladministration is from about 1 mg/day to about 1000 mg/day. In anotheraspect, from about 1 mg/day to about 500 mg/day. In still anotheraspect, from about 1 mg/day to about 100 mg/day. In yet another aspect,from about 0.1 mg/day to about 10 mg/day.

In another aspect, the therapeutically effective amount is administeredin a dosage of between about 1 mg to about 500 mg per dose.

In still another aspect, the therapeutically effective amount comprisesa dosage of between about 1 mg to about 100 mg per dose.

Preferred dosages for administration by inhalation are from about 0.1 pgto about 1 μg per day.

Preferred dosages for administration by injection, i.e., parenteraladministration, are from about 100 ng to about 1 mg per day.

The present invention also includes the use of a compound of Formula(I), or a pharmaceutically acceptable salt thereof for the manufactureof a medicament for use in treating a subject who has, or in preventinga subject from developing, NSP and/or CFP and symptoms associated withthose poisonings, and who is in need of such treatment.

In one aspect, this use of a compound of formula (I) can be employedwhere the disease or condition is chronic obstructive pulmonary disease.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease or condition is asthma.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease or condition is pulmonary disease.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease or condition is pulmonary infection.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease or condition is cystic fibrosis.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease or condition is chronic bronchitis.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease or condition is Karteneger's syndrome.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease or condition is bronchiectasis.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease or condition is an industrial related disease causedor exacerbated by inhaling gases, particles of textiles, grit, or otherindustrial particles or fumes. Specific examples of particles and gritinclude, for example, iron oxides, silica, talc, carbon, graphite,fibers, wood dust, grain dust, organic solvents and pollutant gases.

In still another aspect, the compounds of formula (I) and thepharmaceutical compositions comprising formula (I) can be employed wherethe disease or condition results from inhalation of bacterial or otherpathogenic particles, e.g., fungal particles. Thus, the invention alsoencompasses methods of clearing pathogenic particles, such as particlesthat comprise bacteria, e.g., anthrax or fungus particles.

The present invention also includes a container kit including aplurality of containers, each container including one or more unit doseof a compound of formula (I), or a pharmaceutically acceptable saltthereof.

In an embodiment, this container kit includes each container adapted fororal delivery and includes a tablet, gel, or capsule or inhaler.

In an embodiment, this container kit includes each container adapted forparenteral delivery and includes a depot product, syringe, ampoule, orvial.

In an embodiment, this container kit includes each container adapted fortopical delivery and includes a patch, medipad, ointment, or cream.

The compounds of formula (I) can form salts when reacted withappropriate acids or bases. Pharmaceutically acceptable salts aregenerally preferred over the corresponding compounds of formula (I)since they frequently produce compounds that are usually more watersoluble, stable and/or more crystalline. Pharmaceutically acceptablesalts are any salt which retains the activity of the parent compound anddoes not impart any deleterious or undesirable effect on the subject towhom it is administered and in the context in which it is administered.Pharmaceutically acceptable salts include acid addition salts of bothinorganic and organic acids. Preferred pharmaceutically acceptable saltsinclude salts such as those described by Berge, Bighley, and Monkhouse,J. Pharm. Sci., 1977, 66, 1–19. Such salts may be formed from inorganicand organic acids. Representative examples thereof include maleic,fumaric, benzoic, ascorbic, pamoic, succinic, bismethylenesalicylic,methanesulfonic, ethanedisulfonic, acetic, propionic, tartaric,salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic,glycolic, p-aminobenzoic, glutamic, benzenesulfonic, hydrochloric,hydrobromic, sulfuric, cyclohexylsulfamic, phosphoric and nitric acids.For other acceptable salts, see Int. J. Pharm., 33, 201–217 (1986).

Hydrates and solvates of the compounds along with polymorphs thereof arealso forms of the compounds of the invention and may be formed accordingto techniques known to one having ordinary skill in the pharmaceuticalarts. The invention further includes complexes, particularlyorgano-metallic complexes, of the compounds of the invention. Complexescan be prepared when appropriate using processeses known in the art.

METHODS OF THE INVENTION

The compounds of the invention, pharmaceutical formulations comprisingsaid compounds, and pharmaceutically acceptable salts thereof, areuseful for treating a subject, preferably a mammal, more preferably ahuman, suffering from a disease or condition associated with decreasedmucus clearance, and are useful for helping to prevent or delay theonset of such a disease or condition. The compounds and formulations ofthe invention are particularly useful for treating, preventing, orslowing the progression of chronic obstructive pulmonary disease,asthma, pulmonary disease, pulmonary infection, and cystic fibrosis.When treating or preventing a disease and condition associated withdecreased mucus clearance, and the associated symptoms, the compounds ofthe invention can either be used individually or in combination, as isbest for the subject.

With regard to these diseases and conditions, the term “treating” meansthat compounds of the invention can be used in subjects, preferablyhuman subjects/patients, with existing condition or disease. Thecompounds of the invention will not necessarily cure the subject who hasthe disease but will delay or slow the progression or prevent furtherprogression of the disease thereby giving the individual a more usefullife span.

The term “preventing” means that that if the compounds of the inventionare administered to those who do not now have the disease, or symptom(s)of the condition, but who would normally develop the disease or be atincreased risk for the disease, they will not develop the disease. Inaddition, “preventing” also includes delaying the development of thedisease in an individual who will ultimately develop the disease orwould be at risk for the disease due to age, familial history, geneticor chromosomal abnormalities, and/or due to the presence of one or morebiological markers for the disease. By delaying the onset of thedisease, compounds of the invention can prevent the individual fromgetting the disease during the period in which the individual wouldnormally have gotten the disease or reduce the rate of development ofthe disease or some of its effects but for the administration ofcompounds of the invention up to the time the individual ultimately getsthe disease. Preventing also includes administration of the compounds ofthe invention to those individuals thought to have predisposition forthe disease.

In a preferred aspect, the compounds of the invention are useful forslowing the progression of disease symptoms.

In another preferred aspect, the compounds of the invention are usefulfor preventing the further progression of disease symptoms.

In treating or preventing the above diseases, the compounds of theinvention are administered in a therapeutically effective amount. Thetherapeutically effective amount will vary depending on the particularcompound used, the physical characteristics of the subject to betreated, and the route of administration, as is known to those skilledin the art.

In treating a subject displaying any of the diagnosed above conditions aphysician may administer a compound of the invention immediately andcontinue administration indefinitely, as needed.

Dosage Forms and Amounts

The compounds of the invention can be administered by inhalation,orally, parenterally, (IV, IM, depo-IM, SQ, and depo SQ), sublingually,intranasally, intrathecally, topically, vaginally, or rectally. Dosageforms known to those of skill in the art are suitable for delivery ofthe compounds of the invention.

Compositions are provided that contain therapeutically effective amountsof the compounds of the invention. The compounds are preferablyformulated into suitable pharmaceutical preparations such as tablets,capsules, or elixirs for oral administration or in sterile solutions orsuspensions for parenteral administration. Typically the compoundsdescribed above are formulated into pharmaceutical compositions usingtechniques and procedures well known in the art.

About 1 pg to about 100 mg of a compound or mixture of compounds of theinvention or a physiologically acceptable salt or ester is compoundedwith a physiologically acceptable vehicle, carrier, excipient, binder,preservative, stabilizer, flavor, etc., in a unit dosage form as calledfor by accepted pharmaceutical practice. The amount of active substancein those compositions or preparations is such that a suitable dosage inthe range indicated is obtained. The compositions are preferablyformulated in a unit dosage form, each dosage containing from about 0.1pg to about 100 mg, preferably about 0.1 pg to about 10 mg, morepreferably about 0.1 pg to about 10 pg, or about 1 pg to about 10 mg, ofthe active ingredient. The term “unit dosage from” refers to physicallydiscrete units suitable as unitary dosages for human subjects and othermammals, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient.

To prepare pharmaceutically acceptable compositions of the invention,one or more compounds of the invention are mixed with a suitablepharmaceutically acceptable carrier. Upon mixing or addition of thecompound(s), the resulting mixture may be a solution, suspension,emulsion, or the like. Liposomal suspensions may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forlessening or ameliorating at least one symptom of the disease, disorder,or condition treated and may be empirically determined.

Pharmaceutical carriers or vehicles suitable for administration of thecompounds provided herein include any such carriers known to thoseskilled in the art to be suitable for the particular mode ofadministration. In addition, the active materials can also be mixed withother active materials that do not impair the desired action, or withmaterials that supplement the desired action, or have another action.The compounds may be formulated as the sole pharmaceutically activeingredient in the composition or may be combined with other activeingredients.

Where the compounds exhibit insufficient solubility, methods forsolubilizing may be used. Such methods are known and include, but arenot limited to, using cosolvents such as dimethylsulfoxide (DMSO), usingsurfactants such as Tween®, and dissolution in aqueous sodiumbicarbonate. Derivatives of the compounds, such as salts or prodrugs mayalso be used in formulating effective pharmaceutical compositions.

The concentration of the compound is effective for delivery of an amountupon administration that lessens or ameliorates at least one symptom ofthe disorder for which the compound is administered. Typically, thecompositions are formulated for single dosage administration.

The compounds of the invention may be prepared with carriers thatprotect them against rapid elimination from the body, such astime-release formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, microencapsulateddelivery systems. The active compound is included in thepharmaceutically acceptable carrier in an amount sufficient to exert atherapeutically useful effect in the absence of undesirable side effectson the subject treated. The therapeutically effective concentration maybe determined empirically by testing the compounds in known in vitro andin vivo model systems for the treated disorder.

The compounds and compositions of the invention can be enclosed inmultiple or single dose containers. The enclosed compounds andcompositions can be provided in kits, for example, including componentparts that can be assembled for use. For example, a compound inhibitorin lyophilized form and a suitable diluent may be provided as separatedcomponents for combination prior to use. A kit may include a compoundinhibitor and a second therapeutic agent for co-administration. Theinhibitor and second therapeutic agent may be provided as separatecomponent parts. A kit may include a plurality of containers, eachcontainer holding one or more unit dose of the compound of theinvention. The containers are preferably adapted for the desired mode of(administration, including, but not limited to tablets, gel capsules,sustained-release capsules, and the like for oral administration; depotproducts, pre-filled syringes, ampoules, vials, and the like forparenteral administration; and patches, medipads, creams, and the likefor topical administration.

The concentration of active compound in the drug composition will dependon the route of administration, and the distribution, metabolism, andexcretion rates of the compound, as well as the dosage schedule, andamount administered, together with other factors known to those of skillin the art.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

If oral administration is desired, the compound should be provided in acomposition that protects it from the acidic environment of the stomach.For example, the composition can be formulated in an enteric coatingthat maintains its integrity in the stomach and releases the activecompound in the intestine. The composition may also be formulated incombination with an antacid or other such ingredient.

Oral compositions will generally include an inert diluent or an ediblecarrier and may be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, the activecompound or compounds can be incorporated with excipients and used inthe form of tablets, capsules, or troches. Pharmaceutically compatiblebinding agents and adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches, and the like can contain any ofthe following ingredients or compounds of a similar nature: a bindersuch as, but not limited to, gum tragacanth, acacia, corn starch, orgelatin; an excipient such as microcrystalline cellulose, starch, orlactose; a disintegrating agent such as, but not limited to, alginicacid and corn starch; a lubricant such as, but not limited to, magnesiumstearate; a gildant, such as, but not limited to, colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; and aflavoring agent such as peppermint, methyl salicylate, or fruitflavoring.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials, whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, chewing gum orthe like. A syrup may contain, in addition to the active compounds,sucrose as a sweetening agent and certain preservatives, dyes andcolorings, and flavors.

The active materials can also be mixed or blended with otherpharmaceutically acceptable active agents that do not impair the desiredaction, or with materials that supplement the desired action.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent such as water for injection, saline solution, fixed oil,a naturally occurring vegetable oil such as sesame oil, coconut oil,peanut oil, cottonseed oil, and the like, or a synthetic fatty vehiclesuch as ethyl oleate, and the like, polyethylene glycol, glycerine,propylene glycol, or other synthetic solvent; antimicrobial agents suchas benzyl alcohol and methyl parabens; antioxidants such as ascorbicacid and sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid (EDTA); buffers such as acetates,citrates, and phosphates; and agents for the adjustment of tonicity suchas sodium chloride and dextrose. Parenteral preparations can be enclosedin ampoules, disposable syringes, or multiple dose vials made of glass,plastic, or other suitable material. Buffers, preservatives,antioxidants, and the like can be incorporated as required.

Where administered intravenously, suitable carriers includephysiological saline, phosphate buffered saline (PBS), and solutionscontaining thickening and solubilizing agents such as glucose,polyethylene glycol, polypropyleneglycol, and mixtures thereof.Liposomal suspensions including tissue-targeted liposomes may also besuitable as pharmaceutically acceptable carriers. These may be preparedaccording to methods known for example, as described in U.S. Pat. No.4,522,811.

The active compounds may be prepared with carriers that protect thecompound against rapid elimination from the body, such as time-releaseformulations or coatings. Such carriers include controlled releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers such as collagen, ethylene vinyl acetate, polyanhydrides,polyglycolic acid, polyorthoesters, polylactic acid, and the like.Methods for preparation of such formulations are known to those skilledin the art.

The compounds of the invention can be administered by inhalation (eitherorally or intranasally), orally, parenterally (IV, IM, depo-IM, SQ, anddepo-SQ), sublingually, intrathecally, topically, or rectally. Dosageforms known to those skilled in the art are suitable for delivery of thecompounds of the invention.

Compounds of the invention may be administered enterally orparenterally. When administered orally, compounds of the invention canbe administered in usual dosage forms for oral administration as is wellknown to those skilled in the art. These dosage forms include the usualsolid unit dosage forms of tablets and capsules as well as liquid dosageforms such as solutions, suspensions, and elixirs. When the solid dosageforms are used, it is preferred that they be of the sustained releasetype so that the compounds of the invention need to be administered onlyonce or twice daily.

The oral dosage forms are administered to the subject 1, 2, 3, or 4, ormore or as needed, times daily. It is preferred that the compounds ofthe invention be administered either three or fewer times, morepreferably once or twice daily. Hence, it is preferred that thecompounds of the invention be administered in oral dosage form. It ispreferred that whatever oral dosage form is used, that it be designed soas to protect the compounds of the invention from the acidic environmentof the stomach. Enteric coated tablets are well known to those skilledin the art. In addition, capsules filled with small spheres each coatedto protect from the acidic stomach, are also well known to those skilledin the art.

As noted above, depending on whether asymmetric carbon atoms arepresent, the compounds of the invention can be present as mixtures ofisomers, as racemates, or in the form of pure isomers.

Salts of compounds are preferably the pharmaceutically acceptable ornon-toxic salts of compounds of formula I. For isolation andpurification purposes it is also possible to use pharmaceuticallyunacceptable salts.

EXAMPLES Example 1

Synthesis of Compounds

Brevenal Isolation

Brevenal can be isolated and purified from native sources, such as K.brevis, or other red tide organisms. Suitable purification methodologiesare well known in the art. See, for example, Baden et al, 1981, Toxicon19:455–463; Poli et al., Molecular Pharmacology, 1986 30:129–135. Thefollowing procedure is representative.

Brevenal is extracted from K. brevis cultures (Provasoli—GuillardNational Center for Culture of Marine Phytoplankton, West BoothbayHarbor, Me.) using chloroform. The chloroform layers are collected,dried and partitioned between petroleum ether and aqueous methanol toremove pigments and cellular lipid debris. The aqueous methanol layer(90%) is dried under vacuum and the components separated using a silicagel column (mobile phase CHCl₃:methanol:acetic acid; 100:10:1 v/v).Brevenal and brevetoxins coelute from the silica column; fractionscontaining these materials are collected and combined. A low-pressureC18 matrix column is used to separate the remaining pigments frombrevetoxins and brevenal using an acetonitrile:water mobile phase (80:20v/v), creating a “clarified” extract. The clarified extract is appliedto an HPLC column, such as a Varian C18 reverse-phase column (0.8×25 cm)with a running buffer of 90:10 methanol:water at an appropriate pumprate (e.g., 3–4 mL/min). Detection of eluate peaks can be done by anymethod known in the art, such as UV detection at 215 nm. Peaks ofinterest are isolated and applied to another column, such as ahydrophobic interaction (HI) column (e.g., Phenomenex C18 phenyl-hexylcolumn 0.8×25 cm) in appropriate running buffer (e.g., 99% MeOH:1% H2O).The fractions containing the compound(s) of interest are pooled and thecompound(s) are isolated by any known method such as crystallization,evaporation of solvent (Roto-Vap).

The structure of brevenal was determined using a number of spectroscopicmethods including NMR, Mass spectroscopy and FT-IR. The exact mass ofbrevenal as determined by high resolution Mass Spectroscopy is 656.4043.The primary and stereospecific structure were elucidated using 1-D and2-D NMR spectroscopy in four different solvents.

Various synthetic methodologies can be used to make compounds of theinvention; brevenal is a suitable starting material. Suitablemethodologies are known in the art. Brevenal can be used as a startingmaterial as is or can first be protected or converted to thecorresponding alcohol or carboxylic acid for subsequent elaboration.Representative synthetic procedures for preparing compounds of theinvention from such starting materials are disclosed in, e.g., Mende, T.J., et al., Tetr. Lett., 1990; 31(37):5307–5310; Trainer, V. L., et al.,Molec. Pharm., 1991; 40(6):988–994; Keck, G. E., et al., TetrahedronLett., 1987, 28:139–142; Alvarez, E., et al., Chem. Rev., 1995,95:1953–1980; Rein, et al., 1994: (a) J. Org Chem., 59:2107–2113; (b) J.Org. Chem. 59:2101–2106. Each of these references is incorporated hereinby reference in its entirety. Those skilled in the art will appreciatethat minor modifications can be made to the particular procedures toarrive at compounds of the invention.

Example 1

α-Amiloride Derivative

Amiloride (N-amidino-3,5-diamino-6-chloropyrazinecarboxamide) exists incharged form under physiological conditions as shown below.

Amiloride, Saxitoxin and Tetrodotoxin Structures

Amiloride Functionalization of Brevenal:

Introduction of a guanidine moiety on the brevenal sidechain isperformed as follows. Brevenal is treated with sodium chlorite andsodium dihydrogenphosphate in a solvent mixture oftert-butanol/2-methyl-2-butene to lead to the acid brevenal derivative 2(scheme 2). Treatment of 2 with CDI in DMF followed by addition ofguanidine (free base) or guanidine carbonate affords the guanidinecompound 3.

Preparation of Benzamyl Guanidine Brevenal:

The synthesis is performed by essentially following the same procedureas described for the synthesis of 3 with addition of benzamyl guanidineinstead of guanidine (scheme 2). Alternatively, Brevenal can beconverted into the corresponding acid chloride to effectuate theconversion to the guanidine derivatives.

Example 2

β-Naphthoyl Derivative

β-naphthoyl brevenal is prepared as follows. A solution ofcarbonyldiimidazole and 2-naphthoic acid in benzene is added to brevenoland refluxed overnight to furnish β-naphthoyl brevenal 5 (scheme 3).

Brevenal is treated with sodium borohydride and cerium chloride in amixture of DMF/methanol. After an ether extraction, the crude extract ispurified on HPLC to give brevenol with good yield. Alternatively,Brevenal can first be converted into the corresponding carboxylic acidand reacted subsequently reacted with an appropriate alcohol to affordan ester.

Example 3

Aromatic Brevenal

A. Introduction of the hydroxy benzyl moiety is achieved through aGrignard reaction between phenylmagnesium bromide and brevenal (scheme4). Extraction followed by purification affords an ephedrine adduct ofbrevenal 6.

B. A one step synthesis can be used to prepare several benzeniccompounds. Using a Wittig reaction and commercially availablephosphoranes, various derivatives involving a phenyl group can be made.Thus, treatment of phosphoranes 7a–7f with base followed by addition ofbrevenal furnishes the corresponding ethylenic compounds 8a–bf (scheme5). Compounds may be further purified by HPLC.

Example 4

Benzimidazolone Derivatives

The synthesis begins by the activation of brevenol into a mesylateintermediate followed by the deplacement of the mesylate by iodide toyield to iodo-brevenal derivative 9 (scheme 6). Then treatment of thecommercially available 1,3-dihydro-benzimidazl-2-one with sodium hydridein DMF followed by addition of iodo-brevenal 9 furnishes, afterextraction and purification, the benzimidazolone-brevenal derivative 10.

Example 5

Nucleoside Model of Brevenal

ATP or UTP is treated with dicyclohexylcarbodiimide in DMF to generatethe reactive moiety that is then added to brevenol. The reaction mixtureis heated under reflux and extracted. Purification of the crude extractaffords the nucleotide derivatives 11 and 12 (scheme 7).

Example 6

Airway Merchanics Experimental Protocols

Measurement of Airway Mechanics—Unsedated sheep are restrained in a cartin the prone position with their heads immobilized. After topicalanesthesia of the nasal passages with 2% lidocaine solution, a ballooncatheter is advanced through one nostril into the lower esophagus. Theanimals are intubated with a cuffed endotracheal tube through the othernostril using a flexible fiber optic bronchoscope. Pleural pressure isestimated with the esophageal balloon catheter (filled with one ml ofair) which is positioned 5–10 cm from the gastroesophageal junction. Inthis position the end expiratory pleural pressure ranges between −2 and−5 cm H₂O. Once the balloon is placed, it is secured so that it remainsin position for the duration of the experiment. Lateral pressure in thetrachea is measured with a sidehole catheter (inner dimension, 2.5 mm)advanced through and positioned distal to the tip of the endotrachealtube. Transpulmonary pressure, the difference between tracheal andpleural pressure, is measured with a differential pressure transducercatheter system which shows no phase shift between pressure and flow upto a frequency of 9 Hz. For the measurement of pulmonary resistance(R_(L)), the proximal end of the endotracheal tube is connected to apneumotachograph (Fleisch, Dyna Sciences, Blue Bell, Pa.). The signalsof flow and transpulmonary pressure are recorded on an oscilloscoperecorder which is linked to a computer for on-line calculation of R_(L)from transpulmonary pressure, respiratory volume (obtained by digitalintegration) and flow by the iso-volume technique. Analysis of 5–10breaths is used for the determination of R_(L) (Abraham et al., 1994).

Aerosol Delivery Systems—All aerosols are generated using a disposablemedical nebulizer (Raindrop®, Puritan Bennett, Lenexa, Kans.) thatprovide an aerosol with a mass median aerodynamic diameter of 3.2 μm(geometric SD 1.9) as determined by an Andersen cascade impactor. Thenebulizer is connected to a dosimeter system, consisting of a solenoidvalve and a source of compressed air (20 psi). The output of thenebulizer was directed into a plastic T-piece, one end of which isconnected to the inspiratory port of a Harvard respirator. The solenoidvalve is activated for one second at the beginning of the inspiratorycycle of the respirator. Aerosols were delivered at a tidal volume of500 ml and a rate of 20 breaths per minute (Abraham et al., 1994).

Airway Responsiveness—To assess airway responsiveness, we performcumulative dose response curves to carbachol by measuring R_(L)immediately after inhalation of buffer and after each consecutiveadministration of 10 breaths of increasing concentrations of carbachol(0.25, 0.5, 1.0, 2.0 and 4.0% w/v buffered saline). The provocation testis discontinued when R_(L) increased over 400% from the post-salinevalue or after the highest carbachol concentration has beenadministered. Airway responsiveness is estimated by determining thecumulative carbachol dose in breath units (BU) that increases R_(L) by400% (PC400) by interpolation from the dose response curve. One breathunit (BU) is defined as 1 breath of an aerosol solution containing 1%wt/vol carbachol (Abraham et al., 1994).

Nasal Airway Resistance—Nasal airway resistance (NAR) is measured with amodified mask rhinomanometry technique. The sheep's head is placed in aplexiglass hood with attachments for a faceplate containing apneumotachograph to measure flow and two catheter ports to measure thepressure differential between nose and mouth pressure (Abraham et al.,1998).

Tracheal Mucus Velocity—Sheep are nasally intubated with an endotrachealtube 7.5 cm in diameter shortened by 6 cm., after topical anesthesia ofthe nasal passages with 2% lidocaine solution. The cuff of the tube isplaced just below the vocal cords (verified by fluoroscopy) in order toallow for maximal exposure of the tracheal surface area. TMV is measuredin vivo by a roentgenographic technique. Between 10 and 20 radiopaqueTeflon/bismuth trioxide disks, 1-mm diameter, 0.8-mm thick and 1.8 mg inweight, are insufflated into the trachea via the endotracheal tube. Thecephalad-axial velocities of the individual disks are recorded onvideotape from a portable image intensifier unit. Individual diskvelocities are calculated by measuring the distance traveled by eachdisk during a 1-min observation period. For each run, the mean value ofall individual disk velocities is calculated. A collar containingradiopaque reference markers of known length are worn by the sheep, andused as a standard to correct for magnification effects inherent in thefluoroscopy unit (O'Riordan et al., 1997).

Statistical Analysis—If the data are normally distributed, thenparametric statistics are used; if data do not conform to a normaldistribution, non-parametric statistics are used. The basic statisticaltests include analysis of variance (ANOVA), i.e. one-way ANOVA ortwo-way ANOVA with repeated measures for multipoint analysis, andunpaired or paired t-test for the appropriate single point analysis. Thenon-parametric counterparts of these tests are: a) the Mann-Whitneytest, which is the counterpart of the unpaired t-test; b) Wilcoxon'ssigned ranks test, the counterpart of the paired t-test; c) Friedman'sAnalysis of Variance for related samples, i.e. randomized blockeddesign; d) the Quade test, also a randomized block design test but foruse with small blocks (n≦4); e) the Kruskal-Wallis test, ANOVA forun-related samples; and f) a non-parametric pairwise comparison,analogous to the parametric Newman-Kuels pairwise test. Whereapplicable, linear regression analysis is performed by method of leastsquares, and correlations will be tested for with Spearman's rho test.For all studies, significance is accepted with p<0.05 on a two tailedanalysis (Conover, 1980).

Example 7

Fish Bioassay

Male mosquito fish (n=104) were used in this assay. Fish were placedindividually in 50 mL beakers containing 20 mL water. The test compounds(PbTx-2 and brevenal) were dissolved in EtOH at a concentration of 0.1mg/mL and added to the fish in a total of 200 μL EtOH. The control fishreceived 200 uL EtOH. Fish were exposed to toxin alone (1 μg/mL water),brevenal alone (1 or 2 μg/mL water), or both brevenal (1.0 μg/mL waterand toxin (1.0 μg/mL water) with the brevenal being added 3 min. beforethe toxin. After addition of the differenc compounds the fish weremonitored for 24 h. or until the time of death. Significant differenceswere determined using a two-way Student's t-test.

Fish exposed to PbTx-2 only died within about 7.5 min., while fishexposed to control, or brevenal at 1 or 2 μg/mL did not die after 24 h.Brevenal effectively protected fish from an equal concentration ofPbTx-2, prolonging life by ˜2.5-fold (with fish dying at about 17 min.after exposure). This suggests that brevenal is not toxic atconcentrations effective for brevetoxin antagonist activity.

Example 8

Competitive rat brain synaptosome assays were performed as described inPoli et al., 1986. The data show that tritiated brevetoxin PbTx-2 waseffectively displaced (˜80%) by brevenal and di-O-Me brevenal when thecompounds were added at about 1000× the concentration of the tritiatedPbTx-2. The approximate EC₅₀ for each are: 3.53 nM (K_(i)=1.76 nM) forPbTx-2; 3.69 uM (K_(i)=1.86 μM) for brevenal; 1.35 μM (K_(i)=0.68 μM)for di-O-Me brevenal.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A compound of the Formula:

wherein R is C₁–C₁₂ alkyl, C₂–C₁₂ alkenyl, C₁–C₁₂ alkyl esters, C₁–C₁₂alkyl amides, C₄–C₁₂ alkenyl esters, C₁–C₁₂ alkylaryl esters, C₄–C₁₂alkenylaryl esters, C₄–C₁₂ alkenyl amides, C₁–C₁₂ alkoxy,formylC₁–C₁₂alkyl, formylC₂–C₁₂alkenyl, alkanoylC₁–C₁₂alkyl,alkanoylC₂–C₁₂alkenyl, carboxyC₁–C₁₂alkyl, or carboxyC₂–C₁₂alkenyl,wherein the alkyl and alkenyl groups are optionally substituted with 1–6substituent groups selected from the group consisting of: C₃–C₁₂cycloalkyl, C₃–C₁₂ heterocyclyl, aryl, heteroaryl, C₁–C₆ alkyl, C₁–C₆alkoxy, halogen, C₂–C₆ alkenyl, OH, aryl esters, cycloalkyl esters,cycloalkenyl esters, and

OR₁ is OH or —O(CO)CH₃; R₂ is —CH═CHCH═CH₂, —CH₂-phenyl, or—CH₂-pyridyl, wherein the phenyl and pyridyl groups are optionallysubstituted at each substitutable position with a group that isindependently C₁–C₆ alkyl, C₁–C₆ alkoxy, haloalkyl, haloalkoxy, hydroxy,hydroxyalkyl, halogen, —CO₂H, C₁–C₆ alkoxycarbonyl, —C(O)NH₂,—C(O)NH(C₁–C₆ alkyl), or —C(O)N(C₁–C₆ alkyl) (C₁–C₆ alkyl); orpharmaceutically acceptable salts, solvates, esters, amides, orcombinations thereof.
 2. The compound according to claim 1, wherein R is

wherein Hal is chloro, fluoro, iodo, or bromo; R₃ is selected from H,OH, NH₂, halogen, and NO₂, and Y is selected from CH, N, O, and S. 3.The compound according to claim 1, wherein the compound is selected fromthe group consisting of:


4. A pharmaceutical composition comprising a therapeutically effectiveamount of at least one compound, pharmaceutically acceptable salt,solvate, ester, amide, or isomer of claim 1, and at least onepharmaceutically acceptable carrier, excipient, solvent, adjuvant,diluent, or mixtures thereof.
 5. A pharmaceutical composition of claim4, wherein the pharmaceutical composition is in unit dosage form.
 6. Thepharmaceutical composition in unit dosage form according to claim 5,wherein the compound is present in an amount of about 1 pg to about 100mg.
 7. A compound of claim 1, wherein


8. A compound according to claim 7, wherein R₁ is H.
 9. A compoundaccording to claim 2, wherein R₂ is —CH═CHCH═CH₂.